1. Expression of an aphid-induced barley methyltransferase in Escherichia coli, purification and characterisation of the enzyme by Irene Ingvor Zetterlund

2. Aim  To test the hypothesis that OMT is involved in gramine biosynthesis  To clone the OMT gene into an expression vector with purpose to synthesize the enzyme in E. coli  To characterize the enzyme kinetically

3. Background: Barley (Hordeum vulgare)  Is an important cereal in Sweden  Is cultivated on the large area of arable land, about 400 000 ha  Is used in the malting industry and for livestock feed  All kind of farm animals can be fed on it

4. Background: Bird cherry-oat aphid ( Rhopalosiphum padi )  One of the most serious barley pests  Transmit virus infections, i. a. barley yellow dwarf virus  Phloem-feeding insects, cause little tissue damage  Are perceived by plants as pathogens  Breed by sexual reproduction and parthenogenesis  Overwinter as eggs on its primary host, bird cherry  In summer make use of diverse grasses as secondary hosts, among them barley

5. Background: Plant defence reactions  Alkaloids – a big group of N-containing secondary metabolites, have strong physiological effects in defence against herbivores  Aphids induce pathogen-defence response  Jasmonic acid signalling pathway induces expression of a wide range of defense genes  One of them was identified as an O- methyltransferase gene

6. Background: Plant defence reactions - Gramine  Gramine - indole proto-alkaloid, secondary metabolite in barley and some other species in the grass family Poaceae  Induced in barley upon aphid infestation  Found in epidermis and in mesophyll parenchyma  Missing in the vascular bundles  The higher gramine amount the lower vulnerability of barley to aphids  Synthesized from tryptophan via 3-aminomethylindole  NMT catalyzes SAM-dependent conversion of AMI to MAMI and from MAMI to gramine

7. Background: OMT  One gene, induced by the aphid, is encoding an O- methyltransferase, OMT  It is also induced by the jasmonic acid signalling pathway  OMTs generally –methylate caffeic acid –lead to lignin precursors –or various classes of flavonoids  But not all of barley cultivars had OMT gene in their genome  In the barley varieties missing the gene, gramine was not found either  In all gramine-containing lines OMT was present

8. Hypothesis  The gene, characterized as encoding for an O-methyltransferase acting on caffeic acid, –might actually be encoding an N- methyltransferase, involved in gramine biosynthesis

9. Methods: IMPACT-CN Protein Purification System  IMPACT = intein mediated purification with an affinity chitin-binding tag  A target protein is fused to a self-cleavable intein tag  A chitin-binding domain in intein tag allows purification of the target protein on the chitin column  The intein tag undergoes specific self- cleavage in presence of DTT  The target protein releases from the chitin- bound intein tag

10. Methods  RT-PCR and PCR  Cloning of the target gene into the vector  Transformation of the competent cells  Agarose and SDS-PAGE gel electrophoresis  Western blotting  Bradford microassay for protein quantification  Silica gel thin-layer chromatography (TLC)

11. Materials: Growth and treatment of plants  H. vulgare, variety Lina, susceptible to the aphids  Sown in November 2003  Grown in a growth chamber at 26 o C, long day, (18 h light/6 h darkness)  5-day-old barley plants were harvested  Their green tissue was treated with 45 μM jasmonic acid for 24 hours to induce the OMT- gene

12. Results: Synthesis of the coding region of the OMT gene  The total RNA was isolated from barley green tissue  RNA was reverse transcribed into single- stranded cDNA using the First-Strand Synthesis System for RT-PCR  To amplify the coding region of the OMT gene by PCR primers OMTcloneF and OMTcloneR2 were used  A product of about 1100 bp was visualized by 2% agarose gel electrophoresis

13. Results: Cloning of the OMT gene into the pTYB 12 vector  The plasmid pTYB12, chosen as a vector - allows the fusion of the cleavable intein tag to the N-terminus of a target protein  The plasmid - digested with the restriction nucleases SmaI and NdeI  The DNA fragment - digested with restriction nuclease NdeI  The digested DNA - ligated into the pTYB12 using the BioLabs Quick Ligation Kit

14. Results: Transformation of E. coli DH5α-T1 and screening for recombinants  To amplify the OMT sequence E. coli DH5α-T1 were transformed with the new plasmid pTYB12-OMT  The recombinant cells were selected on Petri dishes with LB medium containing 100 μg/ml ampicillin  96 randomly chosen colonies were inoculated in a microtitre plate in LB/amp medium  PCR test for inserts using intern primers OMT F1 and OMT R1 obtained 3 clones

15. Results: Control of the inserts  To confirm the obtained recombinant clones, digesting reactions with restriction nucleases Kpn I, Nco I, Nde I and Sap I were carried out over night at 37 o C  The digested DNA was analyzed on 1 % agarose gel  Plasmid 1 gave the expected fragment pattern and thus was chosen as the pTYB12-OMT plasmid  The digesting reaction with restriction nucleases resulted in bands as follow: –Kpn I - 6706 and 1801 bp –Nco I - 7380, 680 and 447 bp –Nde I - 8507 bp –Sap I - 7810 and 697 bp

16. Results: Control of the insert  The plasmid was controlled for the right insert by PCR with 3 pairs of primers: OMT clone F and OMT clone R2 (1); OMT F1 and OMT R1 (2), and Intein Forward and T7 Terminator Reverse (3)  Bands of the correct sizes were visible on 2 % agarose gel, lane 1- 1100 bp, 2 - 348 bp and 3 - 1300 bp  To make sure that there was no error in the sequence of the cloned fragment, the plasmid pTYB12-OMT was sequenced at Cybergene  The sequence proved to be identical to the one published earlier

17. Results: Transformation of E. coli ER2566 and screening for recombinants  The E. coli strain ER2566 was provided by Impact-CN as a host strain for the expression of a target gene cloned in the pTYB12 vector  ER2566 have a chromosomal copy of the T7 RNA polymerase gene inserted into the lacZ gene, and therefore under the control of the lacZ promoter  Expression of T7 RNA polymerase is suppressed in the absence of IPTG, by the binding of lac I repressor to the lac promoter  The transcription of the fusion protein takes place when IPTG is accessible  Transformed cells ER2566 were selected on Petri dishes with LB/amp medium  To control the protein induction ER2566 was transformed with the pMYB5 vector

18. Results: Induction of protein expression  Induced with 0,5 mM IPTG at RT O/N  SDS-PAGE analysis showed bands 100 kDa  100 kDa = OMT-intein fusion protein  Positive control - ER2566 transformed with pMYB5 vector  Negative control – –uninduced E1  E2 and E6 chosen to continue the experiment

19. Results: Optimizing of the protein induction conditions  Different conditions were verified: –Induction with 0,5 mM and 1 mM IPTG –Temperature and time:  37 o C, 4 and 6 h  RT, O/N  15 o C, O/N  SDS-PAGE analysis showed the strongest band about 100 kDa for the induction with 1 M IPTG at RT O/N

20. Results: Western Blot  Protein bands were transferred onto PVDF membrane by semi-dry transfer apparatus  Immunoblotting: –primary antibodies - against the chitin binding domain –Secondary antibodies - Goat Anti-Rabbit HRP  The protein was detected using the ECL Plus Western Blotting kit and chemiluminescence in the CCD-camera  The strongest bands of about 100 kDa in lanes 4 – 7  E6 induced at RT with 1 mM IPTG showed the strongest band, conditions were the best for the protein expression.

21. Results: Purification of the target protein  Purified using the IMPACT-CN Protein Purification System  1 l cell culture was induced with 1 mM IPTG at RT O/N  Cells were broken by sonication  Clarified cell extract, obtained by centrifugation, was loaded onto chitin column  Cleavage reaction - started by adding Cleavage Buffer with DTT  The protein was eluted using the Column Buffer  SDS-PAGE analysis showed band ~ 43 kDa corresponding to the purified methyltransferase

22. Results: Purification of the target protein - conditions  Different conditions for the on column cleavage reaction were tested: at 4 o C and RT for 24 and 40 hours  Elution with the Column Buffer containing 0,5 M and 1 M NaCl –The highest protein concentration – elution with 0,5 M NaCl  The protein concentration was measured spectrophotometrically, using Bradford microassay method for protein quantification Table 1 Concentration of the target protein Cleavage reaction conditions Concentration of the target protein, μg/ml Eluant 1Eluant 2Eluant 3 RT, 24 h240,0018,800,00 RT, 40 h162,1231,144,81 4 o C, 24 h 66,100,00

23. Results: Silica gel thin-layer chromatography  For determination of the kinetic parameters of the methyltransferase were used as substrates: – AMI, MAMI and caffeic acid  The methylation reactions were started and stopped by adding of: –Start – SAM+ 3 H-SAM (95+5) –Stop - stop buffer  The methylation products were separated by means of TLC- plates standing in TLC solvent  The regions with the reactions products were scraped from the TLC-plates for liquid scintillation counting

24. Results: Assay of the methyltransferase activity  Methyl­transferase activity was measured by estimation of the amount of 3 H-labelled product produced with methyl- 3 H-SAM  3 H count per minute was calculated into built product per 1 mg protein Incubation time, min 0 min30 min60 min AMI0226,3148 MAMI052,671,2 Lina, AMI0-16,6 Table 2 AMI and MAMI methylation products built per 1 mg protein, pmol/min, development in time Concentration, mM 0,75 mM1,5 mM3 mM AMI37,0318,7121,34 MAMI2,38301,53 Table 3 AMI and MAMI methylation products built per 1 mg protein, pmol/min, relative to the substrates concentration

25. Results: Assay of the methyltransferase activity  The reactions with the enzyme extract from barley green tissue did not show any activity  The reactions with the methyltransferase purified by IMPACT-CN obtained some built product, but the data are questionable. Table 5 Caffeic acid methylation products built per 1 mg protein, pmol/min, development in time pH 30 min60 min Enzyme7,5038,5 Enzyme9,018,00 Lina7,500 Lina9,000

26. Discussion: Transformation of E. coli DH5α-T1  E. coli had difficulties to survive after its uptake of the plasmid with the insert OMT  Few recombinant colonies were obtained and the survivors turned out to have mutations in the OMT sequence  The third transformation resulted in a frameshift mutation  The fourth transformation was succesful

27. Discussion: Purification of the target protein

28. Discussion: Assay of the methyltransferase activity  The methylation of AMI: –highest after 30 min incubation, decreasing later –that contradicts the kinetic development in time as a logarithmic function  The methylation of MAMI: – increases in time –highest after 60 min incubation  An explanation - the scraped samples were contaminated and thus are not trustworthy  The methyltransferase activity was analyzed relatively to AMI and MAMI concentration: –production of MAMI from AMI is inversely proportional to the substrate concentration

29. Discussion: Assay of the methyltransferase activity  Enzyme activity with caffeic acid as substrate - very little activity.  The purified enzyme was going through several freeze-thaw cycles between the first measurement with AMI and MAMI as substrate and those with caffeic acid.  This could have resulted in the loss of enzymatic activity.  These experiments have to be repeated with freshly purified enzyme.

30. Summary  The enzyme exhibit little activity with caffeic acid but did methylate AMI and MAMI  Thus it might be involved in gramine synthesis by methylating AMI and MAMI rather than acting as caffeic acid OMT  Described as an O-methyltransferase, but a sequence similarity with other OMTs is only 40%

31. Conclusion  The enzyme carries out the transfer of a methyl group from S-adenosylmethionine to AMI, methylating it to MAMI and a methyl group from SAM to MAMI, with the formation of gamine, in fact acting as an N-methyltransferase in gramine biosynthesis  This work supports the idea that the methyltransferase gene accession number U54767 should be classified as an NMT-gene involved in gramine biosynthesis